Design and synthesis of new esters of terpenoid alcohols as 15-lipoxygenase inhibitors

Objective(s): 15-Lipoxygenases are one of the iron-containing proteins capable of performing peroxidation of unsaturated fatty acids in animals and plants. The critical role of enzymes in the formation of inﬂammations, sensitivities, and some cancers has been demonstrated in mammals. The importance of enzymes has led to the development of mechanistic studies, product analysis, and synthesis of inhibitors. Materials and Methods: The inhibitory activity of all synthetic compounds against SLO (soybean 15-lipoxygenase: L1; EC 1,13,11,12) was determined using the peroxide formation method. In this method, the basis of evaluation of lipoxygenase activity is measuring the concentration of fatty acid peroxide. All measurements were compared with 4-methyl-2-(4-methylpiperazinyl)pyrimido[4,5-b]benzothiazine (4-MMPB) as one of the known lipoxygenase inhibitors. The radical scavenging ability of all synthetic compounds using stable free radicals (DPPH: 2,2-diphenyl-1-picrylhydrazyl) was measured for further investigation. Results: In this study, a series of esters from phenolic acids with terpenoid alcohols was synthesized and their inhibitory potency against soybean 15-lipoxygenase and their free radical scavenging properties were determined. Among the synthetic compounds, adamantyl protocatetuate 2j and bornyl protocatetuate 2o showed the most potent inhibitory activity with IC50 values of 0.95 and 0.78 μm, respectively. Conclusion: By changing the alcohol and acyl portions of stylosin, it was found that electronic properties play main role in lipoxygenase inhibition potency in contrast with steric features. Insertion of more reductive phenolic moiety such as catechuate and gallate lead to more lipoxygenase inhibition potency of the esters as observed in their radical scavenging activity.


Introduction
Mammalian lipoxygenases are iron-bound enzymes that are responsible for the oxidation of unsaturated fatty acids and esters to hydroproxy derivatives ( Figure  1). Enzymes of this group have been widely found in plants and animals and are also named based on the situation where a key substrate, such as arachidonic acid and or other unsaturated fatty acids containing a cis, cis-1,4-pentadiene moiety, is oxidized (1). Based on the results of recent research, 15-lipoxygenases have been introduced as an interesting target for interfering treatment of some diseases (2). It has been shown that 15-lipoxygenases are effective in the progression of certain cancers (3)(4)(5)(6). In recent years, findings have been discovered based on the inhibitory effect of the 15-lipoxygenase enzymes in cardiovascular disease treatment (7). Researches have shown that products derived from arachidonic acid and linoleic acid oxidation by 15-lipoxygenases can be used as preinflammation and pre-anorexia agents (8). Three main pathways have been identified that inhibit the activity of lipoxygenase: (1) Reducing inhibitors or antioxidants that interact with the reduction of 15-lipoxygenase. 2) Complexing iron-fixing agents, 3) Non-reducing competitive inhibitors, which compete with arachidonic acid or other unsaturated fatty acids containing a cis, cis-1,4-pentadiene moiety in order to bind to the active site of the enzyme (9).
Natural compounds are a source of lipoxygenase enzyme inhibitors (10). Phenolic compounds such as catechols and cinnamic acid derivatives, which have hydroxyl groups, are appropriate inhibitors of lipoxygenases due to their antioxidant properties. Caffeic acid has been shown to act as an inhibitor of the lipoxygenase enzyme (11). Stylosin, which was extracted from ferula plant for the first time, is a natural monoterpenoid compound that consists of two parts: monoteropen (fenchol) and vanillic acid (12). Studies conducted on its inhibitory power against the 15-lipoxygenase enzymes showed that this compound has an appropriate activity. According to this finding, we decided to synthesize a series of stylosin-based ester compounds and investigate their inhibitory activity as well as their free radical scavenging properties.

15-lipoxygenase inhibitory assessment
Linoleic acid and two assay solutions (A and B)

Instrument
Melting points of the products were determined with an Electrothermal Type 9100 melting point apparatus. The FT-IR spectra were recorded on pressed KBr pellets using an AVATAR 370 FT-IR spectrometer (Therma Nicolet spectrometer, USA) at room temperature in the range between 4000 and 400 cm-1 with a resolution of 4 cm-1. The NMR spectra were provided by Brucker Avance 300, 400 and 500 MHz instruments in deuterochloroform (CDCl 3 ) and deuterated dimethyl sulfoxide (DMSO-d 6 ) in the presence of tetramethylsilane as the internal standard, and the coupling constants (J values) are given in Hz. Elemental analyses were performed using a Thermo Finnigan Flash EA 1112. Mass spectra were recorded with a CH7A Varianmat Bremem instrument at 70 eV electron impact ionization, in m/z (rel%).

General Procedure for Preparation of ester compounds (2a-p)
N,N'-Dicyclohexylcarbodiimide (DCC) (2 mmol) was added to the solution of carboxylic acid (1.5 mmol) and   (1.5 mmol) in dry THF (10 ml) at 0 °C . After about 5 min, the solution was stirred overnight at room temperature. The reaction mixture was filtered off, the solvent was removed in vaccum and extracted with ethyl acetate (EtOAc; 3×10 ml), washed with diluted aqueous citric acid solution (20 ml), saturated aqueous NaHCO 3 solution (20 ml) and water (15 ml), dried over MgSO 4 and the crude residue was purified by column chromatography on silica gel (18).

General procedure for preparation of carboxylic amides (3a ' -b ' )
To a solution of compound 2a ' and 2b ' (25 mmol) and triethylamine (25 mmol, 0.28 ml) in chloroform (15 ml), ethylchloroformate (25 mmol, 0.4 ml) was added dropwise at 0 °C . The solution was stirred for 30 min at 0°C. This solution was added to a mixture of amantadine (27 mmol, 3.42 g) and triethyl amine (25 mmol, 0.28 ml) in chloroform (15 ml). The reaction mixture was stirred at room temperature for 30 min. Then, the mixture filtered and the solvent evaporated in vaccum. The residue was extracted with EtOAc (3×30 ml), washed with saturated aqueous NaHCO 3 solution (30 ml), 0.1 M HCl (30 ml) and water (15 ml), dried over MgSO 4 and the solvent evaporated. The residue was dissolved in mixture of K 2 CO 3 (25 mmol, 3.45 g) in methanol (45 ml). The reaction mixture was refluxed for 4 hr. The mixture was neutralized by dilute aqueous HOAc solution and extracted by EtOAc (3×45 ml). The solvent was evaporated in vaccum and the crude product was purified by column chromatography on silica gel (eluent: chloroform/ methanol) (22).

Results
All esters were synthesized through estrification of monoterpenes with phenolic acids in the presence of N,N'-Dicyclohexylcarbodiimide (DCC) in a THF solvent (Figure 3). This method showed a better yield than esterification of acid chloride with alcohol. All phenolic acids and monoterpenes were purchased from commercial sources.
The radical scavenging ability of all synthetic compounds using stable free radicals (DPPH: 2,2-diphenyl-1-picrylhydrazyl) has been previously measured (Table 1) (15). In this assessment, the DPPH bleaching potency of the synthetic compounds has been reported as IC 50 values.

Discussion
The stylosin 2a showed well inhibitory activity against SLO (IC 50 =40.1±2.2 μM). It is an ester of vanillic acid and fenchol (a monoterpene alcohol). A series of changes were made on both terpene and acidic moieties in order to synthesize appropriate inhibitors and study the structural effects on inhibition potency.
First, compound 2d was synthesized by methylation of the hydroxyl group at the para-benzene ring of 2a. The inhibition potency of 2d was very low (IC 50 > 500 μM). Also, 2c with only one methoxy group at para position showed no inhibitory effect (IC 50 > 500 μM).
According to the recent studies on the inhibitory potency of the phenolic acids, it has been found that esters with hydrophobic rings such as borneol have a potent inhibitory effect on the lipoxygenase enzyme (16). Based on this, it was decided to change the monoterpene moiety of the stylosin to synthesize more potent inhibitors.
Compound 2o that was obtained by replacing the fenchol with borneol in compound 2b, showed a better inhibitory activity compared to compound 2b (IC 50 = 0.78 ± 0.11 μM). Compound 2l that was synthesized through the replacement of fenchol with adamantol in the compound 2g showed a relatively lower inhibitory potency compared to compound 2g (IC 50 = 3.9 ± 0.61 μM).
Compound 2m was synthesized by the replacement of fenchol with ''adamantyl methanol'' in compound 2b, which showed a relatively lower inhibitory potency compared to compound 2b (IC 50 = 3.57 ± 0.19 μM), and compound 2n, which was synthesized from esterification of adamantly methanol with caffeic acid, had a relatively lower inhibitory effect than the compound 2g (IC 50 = 2.70 ± 0.13 μM).
Although compound 2m showed less potency for lipoxygenase inhibition in comparison with that of compound 2b, its radical scavenging potency was not different from compound 2b. By investigating the ability of radical scavenging of the synthetic compounds, it was found that compounds containing several hydroxyl groups on the benzene ring were more capable of free radical trapping. Also, by comparing compounds 2b and 2g wherein the compound 2g has an added dual bond to compound 2b, it was found that their free radical scavenging ability is the same as their lipoxygenase inhibitory activity.
Through investigations on the inhibitory effect of amide compounds against the soybean 15-lipoxygenase enzyme, it was proposed that amide compounds may have a better inhibitory effect than their constituents (17). So, it was decided to prepare amide compounds 3a' and 3b' and to further investigate their lipoxygenase inhibitory potency.
Amide compounds 3a' and 3b' were synthesized during the two reaction steps: reaction of amantadine with the phenolic acids protected by acetyl and then the hydrolysis of the acetyl group (Figure 4).
The lipoxygenase inhibitory potency of compound 3a' was much better than stylosin (IC 50 = 5.40 ± 0.55 μM). However, this amide compound showed a lower inhibitory activity compared to compound 2j. Interestingly, the synthetic amides showed better  radical scavenging activity than their steric homolog, which is probably due to weaker electronegativity effect of amide compared to ester portions. Compound 3b' showed similar inhibitory potency compared to its homolog ester 2f (IC 50 = 188.5 ± 0.14 μM).

Conclusion
By changing the alcohol and acyl portions of stylosin, it was found that electric properties play main role in lipoxygenase inhibition potency in contrast with steric features. Insertion of more reductive phenolic moiety such as catechuate and gallate can lead to more lipoxygenase inhibition activity.